Counteracting osmotic imbalance in a sequencing cell

ABSTRACT

A method of analyzing a molecule is disclosed. A lipid bilayer is formed such that it divides a first reservoir characterized by a first reservoir osmolarity from a second reservoir characterized by a second reservoir osmolarity. An electrolyte solution is flowed to the first reservoir that tends to make a first change to a ratio of the first reservoir osmolarity to the second reservoir osmolarity. A voltage is applied across the lipid bilayer, wherein the lipid bilayer is inserted with a nanopore, and wherein a net transfer of ions between the first reservoir and the second reservoir tends to make a second change to the ratio of the first reservoir osmolarity to the second reservoir osmolarity, and wherein the first change to the ratio and the second change to the ratio tends to counter-balance each other.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/630,342 filed Jun. 22, 2017, which claims priority to U.S.Provisional Application No. 62/355,114, filed Jun. 27, 2016, thedisclosures of which are hereby incorporated by reference in theirentireties for all purposes.

BACKGROUND

Nanopore sequencing systems generally use a protein pore in a planarlipid bilayer (PLB) suspended over a well (e.g., a cylindrical well)containing an electrolyte solution, which is also present in a muchlarger exterior reservoir (e.g., above the well). A working electrodeand counter electrode are used to apply an electrical bias across thewell and the exterior reservoir. The PLB extends over the well to bothelectrically and physically seal the well, and the PLB separates thewell from the larger exterior reservoir. While neutral molecules, suchas water and dissolved gases, may pass through the PLB, ions may not. Aprotein pore in the PLB provides a path for ions to be conducted intoand out of the well.

Protein pores such as alpha hemolysin (aHL) are known to preferentiallyconduct either anions or cations and to have unequal conductivity underpositive and negative electrical bias (Noskov et al., (2004) Biophys J.87:2299). Such properties may lead to a net influx from or efflux intothe well, which leads to diffusion of water through the PLB to balancethe electrolyte concentration between the well and the externalreservoir. Such diffusion can cause instability.

BRIEF SUMMARY

One provided method of analyzing a molecule includes forming a lipidbilayer that divides a first reservoir from a second reservoir. Thefirst reservoir has a first reservoir osmolarity, and the secondreservoir has a second reservoir osmolarity. The method further includesflowing an electrolyte solution to the first reservoir, wherein theelectrolyte solution has an electrolyte solution osmolarity that differsfrom the first reservoir osmolarity, thereby making a first change to aratio of the first reservoir osmolarity to the second reservoirosmolarity. The method further includes applying a voltage across thelipid bilayer, wherein the lipid bilayer includes a nanopore, andwherein the voltage causes a net transfer of ions between the firstreservoir and the second reservoir, thereby making a second change tothe ratio of the first reservoir osmolarity to the second reservoirosmolarity. The first change to the ratio and the second change to theratio substantially counterbalance each other.

In some embodiments, the net transfer of ions between the firstreservoir and the second reservoir includes a net efflux of ions fromthe second reservoir to the first reservoir. In some embodiments, thenet efflux of ions from the second reservoir to the first reservoirincreases the ratio of the first reservoir osmolarity to the secondreservoir osmolarity, and flowing the electrolyte solution to the firstreservoir decreases the ratio of the first reservoir osmolarity to thesecond reservoir osmolarity. In some embodiments, the electrolytesolution osmolarity is lower than the second reservoir osmolarity beforethe electrolyte solution is flowed to the first reservoir. In someembodiments, the method further includes progressively reducing theelectrolyte solution osmolarity from an initial electrolyte solutionosmolarity to a final electrolyte solution osmolarity to make the firstchange to the ratio of the first reservoir osmolarity to the secondreservoir osmolarity.

In some embodiments, the net transfer of ions between the firstreservoir and the second reservoir includes a net influx of ions intothe second reservoir from the first reservoir. In some embodiments, thenet influx of ions into the second reservoir from the first reservoirdecreases the ratio of the first reservoir osmolarity to the secondreservoir osmolarity, and flowing the electrolyte solution to the firstreservoir increases the ratio of the first reservoir osmolarity to thesecond reservoir osmolarity. In some embodiments, the electrolytesolution osmolarity is higher than the second reservoir osmolaritybefore the electrolyte solution is flowed to the first reservoir. Insome embodiments, the method further includes progressively increasingthe electrolyte solution osmolarity from an initial electrolyte solutionosmolarity to a final electrolyte solution osmolarity to make the firstchange to the ratio of the first reservoir osmolarity to the secondreservoir osmolarity.

In some embodiments, the method further includes inserting the nanoporeinto the lipid bilayer before the electrolyte solution is flowed to thefirst reservoir. In some embodiments, the method further includesinserting the nanopore into the lipid bilayer after the electrolytesolution is flowed to the first reservoir. In some embodiments, thelipid bilayer spans across the second reservoir, and the first reservoiris external to the second reservoir. In some embodiments, the firstreservoir has a first reservoir volume, the second reservoir has asecond reservoir volume, and the first reservoir volume is larger thanthe second reservoir volume. In some embodiments, the voltage appliedacross the lipid bilayer is an alternating current voltage. In someembodiments, the voltage applied across the lipid bilayer is a directcurrent voltage.

Also provided is a system for analyzing molecules in a sequencing chip,the system including a sequencing chip including an array of cells,wherein each of the cells includes a well. The system further includes areservoir coupled to the sequencing chip. The system further includes aprocessor or a circuitry configured to form a lipid bilayer that dividesthe reservoir from the well of one of the array of cells. The reservoirhas a first reservoir osmolarity, and the well has a second reservoirosmolarity. The processor or circuitry is further configured to flow anelectrolyte solution to the reservoir, wherein the electrolyte solutionhas an electrolyte solution osmolarity that differs from the firstreservoir osmolarity, thereby making a first change to a ratio of thefirst reservoir osmolarity to the second reservoir osmolarity. Theprocessor or circuitry is further configured to apply a voltage acrossthe lipid bilayer, wherein the lipid bilayer includes a nanopore, andwherein the voltage causes a net transfer of ions between the reservoirand the well, thereby making a second change to the ratio of the firstreservoir osmolarity to the second reservoir osmolarity. The firstchange to the ratio and the second change to the ratio substantiallycounterbalance each other.

In some embodiments, the net transfer of ions between the reservoir andthe well includes a net efflux of ions from the well to the reservoir.In some embodiments, the net efflux of ions from the well to thereservoir increases the ratio of the first reservoir osmolarity to thesecond reservoir osmolarity, and flowing the electrolyte solution to thereservoir decreases the ratio of the first reservoir osmolarity to thesecond reservoir osmolarity. In some embodiments, the electrolytesolution osmolarity is lower than the second reservoir osmolarity beforethe electrolyte solution is flowed to the reservoir. In someembodiments, the method further includes progressively reducing theelectrolyte solution osmolarity from an initial electrolyte solutionosmolarity to a final electrolyte solution osmolarity to make the firstchange to the ratio of the first reservoir osmolarity to the secondreservoir osmolarity.

In some embodiments, the net transfer of ions between the reservoir andthe well comprises a net influx of ions into the well from thereservoir. In some embodiments, the net influx of ions into the wellfrom the reservoir decreases the ratio of the first reservoir osmolarityto the second reservoir osmolarity, and flowing the electrolyte solutionto the reservoir increases the ratio of the first reservoir osmolarityto the second reservoir osmolarity. In some embodiments, the electrolytesolution osmolarity is higher than the second reservoir osmolaritybefore the electrolyte solution is flowed to the reservoir. In someembodiments, the method further includes progressively increasing theelectrolyte solution osmolarity from an initial electrolyte solutionosmolarity to a final electrolyte solution osmolarity to make the firstchange to the ratio of the first reservoir osmolarity to the secondreservoir osmolarity y.

In some embodiments, the processor or the circuitry is furtherconfigured to insert the nanopore into the lipid bilayer before theelectrolyte solution is flowed to the reservoir. In some embodiments,the processor or the circuitry is further configured to insert thenanopore into the lipid bilayer after the electrolyte solution is flowedto the reservoir. In some embodiments, the lipid bilayer spans acrossthe well, and the reservoir is external to the well. In someembodiments, the reservoir has a reservoir volume, the well has a wellvolume, and the reservoir volume is larger than the well volume. In someembodiments, the voltage applied across the lipid bilayer is analternating current voltage. In some embodiments, the voltage appliedacross the lipid bilayer is a direct current voltage.

A better understanding of the nature and advantages of embodiments ofthe present invention may be gained with reference to the followingdetailed description and the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip.

FIG. 2 illustrates an embodiment of a cell 200 performing nucleotidesequencing with the Nano-SBS technique.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags.

FIG. 5 illustrates an embodiment of a cell 500 in a nanopore basedsequencing chip.

FIG. 6A illustrates that initially at time t₁ a nanopore 606 is insertedinto a planar lipid bilayer 604 spanning across a well 602 in a cell ofthe nanopore based sequencing chip.

FIG. 6B illustrates that later at time t₂, as a voltage is appliedacross the lipid bilayer, an osmotic imbalance occurs between theelectrolyte solution above and below the lipid bilayer.

FIG. 6C illustrates that later at time t₃ a net flow of water across thelipid bilayer places a strain on the lipid bilayer, causing the lipidbilayer to rupture or change its shape to a point at which it fails towork, or causing the nanopore to leave the lipid bilayer.

FIG. 7A illustrates that initially at time t₁ a nanopore 706 is insertedinto a planar lipid bilayer 704 spanning across a well 702 in a cell ofthe nanopore based sequencing chip.

FIG. 7B illustrates that later at time t₂, as a voltage is appliedacross the lipid bilayer, an osmotic imbalance occurs between theelectrolyte solution above and below the lipid bilayer.

FIG. 7C illustrates that later at time t₃ a net flow of water across thelipid bilayer and the resulting increase in volume of water in the wellpush the lipid bilayer outward, causing a change in the shape of thelipid bilayer to a point at which the lipid bilayer fails to work.

FIG. 8 is a flowchart of a process 800 for an improved technique ofextending the lifetime of a nanopore inserted in a lipid bilayer in acell of a nanopore based sequencing chip for analyzing molecules.

FIG. 9 illustrates the top view of a nanopore based sequencing system900 with an improved flow chamber enclosing a silicon chip that allowsliquids and gases to pass over and contact sensors on the chip surface.

FIG. 10 is a flowchart of a process 1000 for forming the lipid bilayersin the nanopore based sequencing chip.

FIG. 11A illustrates that by flowing over a lipid bilayer a lowerconcentration of electrolyte solution than is initially present in thewell while the planar lipid bilayer is in place between the well and theexternal reservoir, excess water is forced into the well, causing theplanar lipid bilayer to bow upwards.

FIG. 11B illustrate that at time t₂ the excess volume of water that waspreviously forced into the well due to the initial flow of lowerconcentration electrolyte allows for a greater volume of water to beremoved from the well before the planar lipid bilayer ruptures

FIG. 11C illustrate that at time t₂ for a comparative method, volume ofwater removed from the well forces the nanopore to exit the planar lipidbilayer.

FIG. 12A illustrates the average nanopore lifetime for a comparativemethod in which the external reservoir and well osmolarities are both300 mM.

FIG. 12B illustrates the average nanopore lifetime for a method inaccordance with an embodiment in which the external reservoir osmolarityis 300 mM and the well osmolarity is 340 mM.

FIG. 12C illustrates the average nanopore lifetime for a method inaccordance with an embodiment in which the external reservoir osmolarityis 300 mM and the well osmolarity is 360 mM.

FIG. 13A illustrates an embodiment of a circuitry 1300 in a cell of ananopore based sequencing chip, wherein the circuitry can be configuredto detect whether a lipid bilayer is formed in the cell without causingan already formed lipid bilayer to break down.

FIG. 13B illustrates the same circuitry 1300 in a cell of a nanoporebased sequencing chip as that shown in FIG. 13A. Comparing to FIG. 13A,instead of showing a lipid membrane/bilayer between the workingelectrode and the counter electrode, an electrical model representingthe electrical properties of the working electrode and the lipidmembrane/bilayer is shown.

FIG. 14 shows sets of data points for a bright mode of one cycle and fora dark mode of one cycle.

FIG. 15 shows a block diagram of an example computer system usable withsystems and methods according to embodiments.

TERMS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. Methods, devices, and materials similar or equivalentto those described herein can be used in the practice of disclosedtechniques. The following terms are provided to facilitate understandingof certain terms used frequently and are not meant to limit the scope ofthe present disclosure. Abbreviations used herein have theirconventional meaning within the chemical and biological arts.

“Nanopore” refers to a pore, channel or passage formed or otherwiseprovided in a membrane. A membrane can be an organic membrane, such as alipid bilayer, or a synthetic membrane, such as a membrane formed of apolymeric material. The nanopore can be disposed adjacent or inproximity to a sensing circuit or an electrode coupled to a sensingcircuit, such as, for example, a complementary metal oxide semiconductor(CMOS) or field effect transistor (FET) circuit. In some examples, ananopore has a characteristic width or diameter on the order of 0.1nanometers (nm) to about 1000 nm. Some nanopores are proteins.

“Osmolarity”, also known as osmotic concentration, refers to a measureof solute concentration. Osmolarity measures the number of osmoles ofsolute particles per unit volume of solution. An osmole is a measure ofthe number of moles of solute that contribute to the osmotic pressure ofa solution. Osmolarity allows the measurement of the osmotic pressure ofa solution and the determination of how the solvent will diffuse acrossa semipermeable membrane (osmosis) separating two solutions of differentosmotic concentration.

“Osmolyte” as used herein refers to any soluble compound that whendissolved into a solution increases the osmolarity of that solution.

“Polymerase” refers to an enzyme that performs template-directedsynthesis of polynucleotides. The term encompasses both a full lengthpolypeptide and a domain that has polymerase activity. DNA polymerasesare well-known to those skilled in the art, and include but are notlimited to DNA polymerases isolated or derived from Pyrococcus furiosus,Thermococcus litoralis, and Thermotoga maritime, or modified versionsthereof. They include both DNA-dependent polymerases and RNA-dependentpolymerases such as reverse transcriptase. At least five families ofDNA-dependent DNA polymerases are known, although most fall intofamilies A, B and C. There is little or no sequence similarity among thevarious families. Most family A polymerases are single chain proteinsthat can contain multiple enzymatic functions including polymerase, 3′to 5′ exonuclease activity and 5′ to 3′ exonuclease activity. Family Bpolymerases typically have a single catalytic domain with polymerase and3′ to 5′ exonuclease activity, as well as accessory factors. Family Cpolymerases are typically multi-subunit proteins with polymerizing and3′ to 5′ exonuclease activity. In E. coli, three types of DNApolymerases have been found, DNA polymerases I (family A), II (familyB), and III (family C). In eukaryotic cells, three different family Bpolymerases, DNA polymerases α, δ, and ε, are implicated in nuclearreplication, and a family A polymerase, polymerase γ, is used formitochondrial DNA replication. Other types of DNA polymerases includephage polymerases. Similarly, RNA polymerases typically includeeukaryotic RNA polymerases I, II, and III, and bacterial RNA polymerasesas well as phage and viral polymerases. RNA polymerases can beDNA-dependent and RNA-dependent.

“Nucleic acid” can refer to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. The term canencompass nucleic acids containing known nucleotide analogs or modifiedbackbone residues or linkages, which are synthetic, naturally occurring,and non-naturally occurring, which have similar binding properties asthe reference nucleic acid, and which are metabolized in a mannersimilar to the reference nucleotides. Examples of such analogs caninclude, without limitation, phosphorothioates, phosphoramidites, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

“Template” refers to a strand of a nucleic acid from which acomplementary nucleic acid strand is synthesized by a DNA polymerase,for example, in a primer extension reaction.

“Nucleotide,” in addition to referring to the naturally occurringribonucleotide or deoxyribonucleotide monomers, can be understood torefer to related structural variants thereof, including derivatives andanalogs, that are functionally equivalent with respect to the particularcontext in which the nucleotide is being used (e.g., hybridization to acomplementary base), unless the context clearly indicates otherwise.

“Tag” refers to a detectable moiety that can be atoms or molecules, or acollection of atoms or molecules. A tag can provide an optical,electrochemical, magnetic, or electrostatic (e.g., inductive,capacitive) signature, which signature may be detected with the aid of ananopore. Typically, when a nucleotide is attached to the tag it iscalled a “Tagged Nucleotide.” The tag can be attached to the nucleotidevia the phosphate moiety.

“Substantially counterbalance” as used herein refers to a relationshipbetween two or more changes to an initial value or state, the net effectof which is a change to a value or state that differs from the initialvalue or state by 60% or less. The initial value can be, for example,the ratio of the osmolarity values of two different solutions, or thevolume of liquid within a reservoir. Two changes to the initial valuecan be considered to substantially counterbalance each other if the neteffect of the changes results in a new value (i.e., a new ratio ofosmolarity values) that is 60% less than, 55% less than, 50% less than,45% less than, 40% less than, 35% less than, 30% less than, 25% lessthan, 20% less than, 18% less than, 16% less than, 14% less than, 12%less than, 10% less than. 8% less than, 6% less than, 4% less than, 2%less than, identical to, 2% greater than, 4% greater than, 6% greaterthan, 8% greater than, 10% greater than, 12% greater than, 14% greaterthan, 16% greater than, 18% greater than, 20% greater than, 25% greaterthan, 30% greater than, 35% greater than, 40% greater than, 45% greaterthan, 50% greater than, 55% greater than, or 60% greater than theinitial value. As another example, two changes can substantiallycounterbalance one another if the value of the differences between thetwo changes, divided by the value of either the first or second change,is less than 60%.

Alternatively, two changes to an initial value or state can beconsidered to substantially counterbalance one another if the larger ofthe two changes has a magnitude that is no greater than 60% larger thanthe magnitude of the smaller of the two changes. For example, the largerchange can have a magnitude that is no more than 160% of, no more than150% of, no more than 140% of, no more than 130% of, no more than 120%of, no more than 110% of, no more than 108% of, no more than 106%, nomore than 104% of, or no more than 102% of the magnitude of the smallerchange.

DETAILED DESCRIPTION

Techniques disclosed herein relate to nanopore-based DNA sequencing, andmore specifically, to the use of osmotic imbalance to increase thestability and longevity of nanopores in sequencing cells. Embodimentscan utilize osmolarity imbalance to modulate the time at which a bilayerenters a state that either causes pore ejection or bilayer failure. Inthis manner, embodiments can delay (or prevent premature) pore ejectionor bilayer failure. Such techniques can help maintain a more constantvolume on either side (cis and trans side) of a lipid bilayer containinga nanopore.

Example nanopore systems, circuitry, and sequencing operations areinitially described, followed by example techniques to increase theuseful lifespan of nanopores in DNA sequencing cells. The invention canbe implemented in numerous ways, including as a process; an apparatus; asystem; a composition of matter; a computer program product embodied ona computer readable storage medium; and/or a processor, such as aprocessor configured to execute instructions stored on and/or providedby a memory coupled to the processor.

I. Nanopore System

A. Nanopore Sequencing Cell

Nanopore membrane devices having pore sizes on the order of onenanometer in internal diameter have shown promise in rapid nucleotidesequencing. When a voltage potential is applied across a nanoporeimmersed in a conducting fluid, a small ion current attributed to theconduction of ions through the nanopore can be observed. The size of thecurrent is sensitive to the pore size.

A nanopore based sequencing chip may be used for nucleic acid (e.g.,DNA) sequencing. A nanopore based sequencing chip incorporates a largenumber of sensor cells configured as an array. For example, an array ofone million cells can include 1000 rows by 1000 columns of cells.

FIG. 1 illustrates an embodiment of a cell 100 in an array of cells thatform a nanopore based sequencing chip. A membrane 102 is formed over thesurface of the cell. In some embodiments, membrane 102 is a lipidbilayer. The bulk electrolyte 114 containing soluble protein nanoporetransmembrane molecular complexes (PNTMC) and the analyte of interest(e.g., a single polymer molecule, such as DNA) can be placed directlyonto the surface of the cell. A single PNTMC 104 can be inserted intomembrane 102 by electroporation. The individual membranes in the arrayare neither chemically nor electrically connected to each other. Thus,each cell in the array is an independent sequencing machine, producingdata unique to the single polymer molecule associated with the PNTMC.PNTMC 104 can modulate the ionic current through the otherwiseimpermeable bilayer.

Analog measurement circuitry 112 is connected to a working electrode 110(e.g., made of metal) covered by a volume of electrolyte 108 inside awell formed in an oxide layer 106. The volume of electrolyte 108 isisolated from the bulk electrolyte 114 by the ion-impermeable membrane102. PNTMC 104 crosses membrane 102 and provides the only path for ioniccurrent to flow from the bulk liquid to working electrode 110. The cellalso includes a counter electrode (CE) 116. The cell can also include areference electrode 117, which can act as an electrochemical potentialsensor.

FIG. 5 illustrates an embodiment of a cell 500 in a nanopore basedsequencing chip. Cell 500 includes a well 505 having two side walls anda bottom. In one embodiment, each side wall comprises a dielectric layer504 and the bottom comprises a working electrode 502. In one embodiment,the working electrode 502 has a top side and a bottom side. In anotherembodiment, the top side of 502 makes up the bottom of the well 505while the bottom side of 502 is in contact with dielectric layer 501. Inanother embodiment, the dielectric layer 504 is above dielectric layer501. Dielectric layer 504 forms the walls surrounding a well 505 inwhich a working electrode 502 is located at the bottom. Suitabledielectric materials for use in the present invention (e.g., dielectriclayer 501 or 504) include, without limitation, porcelain (ceramic),glass, mica, plastics, oxides, nitrides (e.g., silicon mononitride orSiN), silicon oxynitride, metal oxides, metal nitrides, metal silicates,transition-metal oxides, transition-metal nitrides, transitionmetal-silicates, oxynitrides of metals, metal aluminates, zirconiumsilicate, zirconium aluminate, hafnium oxide, insulating materials(e.g., polymers, epoxies, photoresist, and the like), or combinationsthereof. Those of ordinary skill in the art will appreciate otherdielectric materials that are suitable for use in the present invention.

As shown in FIG. 5, nanopore cell 500 can be formed on a substrate 530,such as a silicon substrate. Dielectric layer 501 may be formed onsubstrate 530. Dielectric material used to form dielectric layer 501 mayinclude, for example, glass, oxides, nitrides, and the like. An electriccircuit 522 for controlling electrical stimulation and for processingthe signal detected from nanopore cell 500 can be formed on substrate530 and/or within dielectric layer 501. For example, a plurality ofpatterned metal layers (e.g., metal 1 to metal 6) can be formed indielectric layer 501, and a plurality of active devices (e.g.,transistors) can be fabricated on substrate 530. In some embodiments,signal source 528 is included as a part of electric circuit 522.Electric circuit 522 can include, for example, amplifiers, integrators,analog-to-digital converters, noise filters, feedback control logic,and/or various other components. Electric circuit 522 can be furthercoupled to a processor 524 that is coupled to a memory 526, whereprocessor 524 can analyze the sequencing data to determine sequences ofthe polymer molecules that have been sequenced in the array.

In one aspect, cell 500 also includes one or more hydrophobic layers. Asshown in FIG. 5, each dielectric layer 504 has a top surface. In oneembodiment, the top surface of each dielectric layer 504 may comprise ahydrophobic layer. In one embodiment, silanization forms a hydrophobiclayer 520 above the top surface of dielectric layer 504. For example,further silanization with silane molecules that are (i) containing 6 to20 carbon-long chains (e.g., octadecyl-trichlorosilane,octadecyl-trimethoxysilane, or octadecyl-triethoxysilane), (ii)dimethyloctylchlorosilane (DMOC), or (iii) organofunctional alkoxysilanemolecules (e.g., dimethylchloro-octodecyl-silane,methyldichloro-octodecyl-silane, trichloro-octodecyl-silane,trimethyl-octodecyl-silane, or triethyl-octodecyl-silane) can be done onthe top surface of dielectric layer 504. In one embodiment, thehydrophobic layer is a silanized layer or silane layer. In oneembodiment, the silane layer can be one molecule in thickness. In oneaspect, dielectric layer 504 comprises a top surface suitable foradhesion of a membrane (e.g., a lipid bilayer comprising a nanopore). Inone embodiment, the top surface suitable for adhesion of a membranecomprises a silane molecule as described herein. Alternatively, thehydrophobic layer can be a polyimide layer, which is also a dielectric.Polyimide materials have thermal stability, good chemical resistance,and excellent mechanical properties. In some embodiments, hydrophobiclayer 520 has a thickness provided in a nanometer (nM) or micrometer(μm) scale. In other embodiments, the hydrophobic layer may extend downalong all or a part of the dielectric layer 504 (see also Davis et al.U.S. 20140034497, which is incorporated herein by reference in itsentirety).

In another aspect, well 505 (formed by the dielectric layer walls 504)further includes a volume of salt solution 506 above working electrode502. In general, the methods of the present invention comprise the useof a solution (e.g., a salt solution, salt buffer solution, electrolyte,electrolyte solution, or bulk electrolyte) that comprises osmolytes. Inthe present invention, an osmolyte is a compound that is soluble insolution within the architecture of a nanopore sequencing system, e.g.,a well containing a salt solution or a bulk electrolyte as describedherein. As such, the osmolytes of the present invention affect osmosis,particularly osmosis across a lipid bilayer. Osmolytes for use in thepresent invention include, without limitation, ionic salts such aslithium chloride (LiCl), sodium chloride (NaCl), potassium chloride(KCl), lithium glutamate, sodium glutamate, potassium glutamate, lithiumacetate, sodium acetate, potassium acetate, calcium chloride (CaCl₂),strontium chloride (SrCl₂), manganese chloride (MnCl₂), and magnesiumchloride (MgCl₂); polyols and sugars such as glycerol, erythritol,arabitol, sorbitol, mannitol, xylitol, mannisidomannitol, glycosylglycerol, glucose, fructose, sucrose, trehalose, and isofluoroside;polymers such as dextrans, levans, and polyethylene glycol; and someamino acids and derivatives thereof such as glycine, alanine,alpha-alanine, arginine, proline, taurine, betaine, octopine, glutamate,sarcosine, y-aminobutyric acid, and trimethylamine N-oxide (“TMAO”) (seealso e.g., Fisher et al. U.S. 20110053795, incorporated herein byreference in its entirety). In one embodiment, the present inventionutilizes a solution comprising an osmolyte, wherein the osmolyte is anionic salt. Those of ordinary skill in the art will appreciate othercompounds that are suitable osmolytes for use in the present invention.In another aspect, the present invention provides solutions comprisingtwo or more different osmolytes. In some embodiments, the film of saltsolution 506 has a thickness of about three microns (μm).

The architecture of the nanopore based sequencing chip described hereincomprises an array of wells (e.g., FIG. 5) having various volumecapacities, including nanoliter (nL), picoliter (pL), femtoliter (fL),attoliter (aL), zeptoliter (zL) and yocoliter (yL) capacities. Forexample, the volume of electrolyte 108 (e.g., FIG. 1) or salt solution506 (e.g., FIG. 5) is provided in a nL, pL, fL, aL, zL, or yL scale. Inone embodiment of the present invention, the volume of the electrolyteor salt solution formed by the wells (e.g., well 505 in FIG. 5) of thepresent invention, or the volume of electrolyte or salt solution used inmethods described herein may be provided in a nanoliter (nL), picoliter(pL), femtoliter (fL), attoliter (aL), zeptoliter (zL), or yocoliter(yL) scale. The wells may alternately be described by their volume incubic micrometers, or similar dimensions, rather than by volume. It willbe within the ability of one skilled in the art to determine thenecessary conversion between units, for example from cubic micrometersto picoliters, femtoliters, or the like.

As shown in FIG. 5, a membrane is formed on the top surfaces ofdielectric layer 504 and spans across well 505. For example, themembrane includes a lipid monolayer 518 formed on top of hydrophobiclayer 520. As the membrane reaches the opening of well 505, the lipidmonolayer transitions to a lipid bilayer 514 that spans across theopening of the well. The lipid monolayer 518 may also extend along allor a part of the vertical surface (i.e., side wall) of a dielectriclayer 504. In one embodiment, the vertical surface 504 along which themonolayer 518 extends comprises a hydrophobic layer. A bulk electrolyte508 containing protein nanopore transmembrane molecular complexes(PNTMC) and the analyte of interest is placed directly above the well. Asingle PNTMC/nanopore 516 is inserted into lipid bilayer 514. In oneembodiment, insertion into the bilayer is by electroporation. Nanopore516 crosses lipid bilayer 514 and provides the only path for ionic flowfrom bulk electrolyte 508 to working electrode 502. Bulk electrolyte 508can further include one of the following: lithium chloride (LiCl),sodium chloride (NaCl), potassium chloride (KCl), lithium glutamate,sodium glutamate, potassium glutamate, lithium acetate, sodium acetate,potassium acetate, calcium chloride (CaCl₂), strontium chloride (SrCl₂),manganese chloride (MnCl₂), and magnesium chloride (MgCl₂).

Cell 500 includes a counter electrode (CE) 510, which is in electricalcontact with the bulk electrolyte 508. Cell 500 may optionally include areference electrode 512. In some embodiments, counter electrode 510 isshared between a plurality of cells, and is therefore also referred toas a common electrode. The common electrode can be configured to apply acommon potential to the bulk liquid in contact with the nanopores in themeasurements cells. The common potential and the common electrode arecommon to all of the measurement cells.

In some embodiments, working electrode 502 is a metal electrode. Fornon-faradaic conduction, working electrode 502 may be made of metalsthat are resistant to corrosion and oxidation, e.g., platinum, gold,titanium nitride and graphite. For example, working electrode 502 may bea platinum electrode with electroplated platinum. In another example,working electrode 502 may be a titanium nitride (TiN) working electrode.

As shown in FIG. 5, nanopore 516 is inserted into the planar lipidbilayer 514 suspended over well 505. An electrolyte solution is presentboth inside well 505, i.e., trans side, (see salt solution 506) and in apotentially much larger external reservoir 522, i.e., cis side, (seebulk electrolyte 508). The bulk electrolyte 508 in external reservoir522 can be above multiple wells of the nanopore based sequencing chip.Lipid bilayer 514 extends over well 505 and transitions to lipidmonolayer 518 where the monolayer is attached to hydrophobic layer 520.This geometry both electrically and physically seals well 505 andseparates the well from the larger external reservoir. While neutralmolecules, such as water and dissolved gases, can pass through lipidbilayer 514, ions cannot. Nanopore 516 in lipid bilayer 514 provides asingle path for ions to be conducted into and out of well 505.

For nucleic acid sequencing, a polymerase is attached to nanopore 516. Atemplate of DNA is held by the polymerase. The polymerase synthesizesDNA by incorporating hexaphosphate mono-nucleotides (HMN) from solutionthat are complementary to the template. A unique, polymeric tag isattached to each HMN. During incorporation, the tag threads the nanoporeaided by an electric field gradient produced by the voltage betweencounter electrode 510 and working electrode 502. The tag partiallyblocks nanopore 516, procuring a measurable change in the ionic currentthrough nanopore 516. In some embodiments, an alternating current (AC)bias or direct current (DC) voltage is applied between the electrodes.

B. Nanopore-Based Sequencing by Synthesis

In some embodiments, a nanopore array enables parallel sequencing usingthe single molecule nanopore-based sequencing by synthesis (Nano-SBS)technique. FIG. 2 illustrates an embodiment of a cell 200 performingnucleotide sequencing with the Nano-SBS technique. In the Nano-SBStechnique, a template 202 to be sequenced and a primer are introduced tocell 200. To this template-primer complex, four differently taggednucleotides 208 are added to the bulk aqueous phase. As the correctlytagged nucleotide is complexed with the polymerase 204, the tail of thetag is positioned in the barrel of nanopore 206. The tag held in thebarrel of nanopore 206 generates a unique ionic blockade signal 210,thereby electronically identifying the added base due to the tags'distinct chemical structures.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags. A nanopore 301 is formed in a membrane302. An enzyme 303 (e.g., a polymerase, such as a DNA polymerase) isassociated with the nanopore. In some cases, polymerase 303 iscovalently attached to nanopore 301. Polymerase 303 is associated with anucleic acid molecule 304 to be sequenced. In some embodiments, thenucleic acid molecule 304 is circular. In some cases, nucleic acidmolecule 304 is linear. In some embodiments, a nucleic acid primer 305is hybridized to a portion of nucleic acid molecule 304. Polymerase 303catalyzes the incorporation of nucleotides 306 onto primer 305 usingsingle stranded nucleic acid molecule 304 as a template. Nucleotides 306comprise tag species (“tags”) 307.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags. Stage A illustrates the components asdescribed in FIG. 3. Stage C shows the tag loaded into the nanopore. A“loaded” tag may be one that is positioned in and/or remains in or nearthe nanopore for an appreciable amount of time, e.g., 0.1 millisecond(ms) to 10,000 ms. In some cases, a tag that is pre-loaded is loaded inthe nanopore prior to being released from the nucleotide. In someinstances, a tag is pre-loaded if the probability of the tag passingthrough (and/or being detected by) the nanopore after being releasedupon a nucleotide incorporation event is suitably high, e.g., 90% to99%.

At stage A, a tagged nucleotide (one of four different types: A, T, G,or C) is not associated with the polymerase. At stage B, a taggednucleotide is associated with the polymerase. At stage C, the polymeraseis docked to the nanopore. The tag is pulled into the nanopore duringdocking by an electrical force, such as a force generated in thepresence of an electric field generated by a voltage applied across themembrane and/or the nanopore.

Some of the associated tagged nucleotides are not base paired with thenucleic acid molecule. These non-paired nucleotides typically arerejected by the polymerase within a time scale that is shorter than thetime scale for which correctly paired nucleotides remain associated withthe polymerase. Since the non-paired nucleotides are only transientlyassociated with the polymerase, process 400 as shown in FIG. 4 typicallydoes not proceed beyond stage D. For example, a non-paired nucleotide isrejected by the polymerase at stage B or shortly after the processenters stage C.

In various embodiments, before the polymerase is docked to the nanopore,the conductance of the nanopore is ˜300 picosiemens (300 pS). At stageC, the conductance of the nanopore is about 60 pS, 80 pS, 100 pS, or 120pS, corresponding to one of the four types of tagged nucleotidesrespectively. The polymerase undergoes an isomerization and atransphosphorylation reaction to incorporate the nucleotide into thegrowing nucleic acid molecule and release the tag molecule. Inparticular, as the tag is held in the nanopore, a unique conductancesignal (e.g., see signal 210 in FIG. 2) is generated due to the tag'sdistinct chemical structures, thereby identifying the added baseelectronically. Repeating the cycle (i.e., stage A through E or stage Athrough F) allows for the sequencing of the nucleic acid molecule. Atstage D, the released tag passes through the nanopore.

In some cases, tagged nucleotides that are not incorporated into thegrowing nucleic acid molecule will also pass through the nanopore, asseen in stage F of FIG. 4. The unincorporated nucleotide can be detectedby the nanopore in some instances, but the method provides a means fordistinguishing between an incorporated nucleotide and an unincorporatednucleotide based at least in part on the time for which the nucleotideis detected in the nanopore. Tags bound to unincorporated nucleotidespass through the nanopore quickly and are detected for a short period oftime (e.g., less than 10 ms), while tags bound to incorporatednucleotides are loaded into the nanopore and detected for a long periodof time (e.g., at least 10 ms).

Further details regarding the nanopore-based sequencing can be found in,for example, U.S. patent application Ser. No. 14/577,511 entitled“Nanopore-Based Sequencing With Varying Voltage Stimulus,” U.S. patentapplication Ser. No. 14/971,667 entitled “Nanopore-Based Sequencing WithVarying Voltage Stimulus,” U.S. patent application Ser. No. 15/085,700entitled “Non-Destructive Bilayer Monitoring Using Measurement OfBilayer Response To Electrical Stimulus,” and U.S. patent applicationSer. No. 15/085,713 entitled “Electrical Enhancement Of BilayerFormation.”

II. Measurement Circuitry

FIG. 13A shows a lipid membrane or lipid bilayer 1312 situated between acell working electrode 1314 and a counter electrode 1316, such that avoltage is applied across lipid membrane/bilayer 1312. A lipid bilayeris a thin membrane made of two layers of lipid molecules. A lipidmembrane is a membrane having a thickness of several molecules (morethan two) of lipid molecules. Lipid membrane/bilayer 1312 is also incontact with a bulk liquid/electrolyte 1318. Note that working electrode1314, lipid membrane/bilayer 1312, and counter electrode 1316 are drawnupside down as compared to the working electrode, lipid bilayer, andcounter electrode in FIG. 1. In some embodiments, the counter electrodeis shared between a plurality of cells, and is therefore also referredto as a common electrode. The common electrode can be configured toapply a common potential to the bulk liquid in contact with the lipidmembranes/bilayers in the measurements cells by connecting the commonelectrode to a voltage source V_(liq) 1320. The common potential and thecommon electrode are common to all of the measurement cells. There is aworking cell electrode within each measurement cell; in contrast to thecommon electrode, working cell electrode 1314 is configurable to apply adistinct potential that is independent from the working cell electrodesin other measurement cells.

FIG. 13B illustrates the same circuitry 1300 in a cell of a nanoporebased sequencing chip as that shown in FIG. 13A. Comparing to FIG. 13A,instead of showing a lipid membrane/bilayer between the workingelectrode and the counter electrode, an electrical model representingthe electrical properties of the working electrode and the lipidmembrane/bilayer is shown.

Electrical model 1322 includes a capacitor 1324 representing theelectrical properties of working electrode 1314. The capacitanceassociated with working electrode 1314 is also referred to as a doublelayer capacitance (C_(double layer)). Electrical model 1322 furtherincludes a capacitor 1326 (C_(bilayer)) that models a capacitanceassociated with the lipid membrane/bilayer and a resistor 1328(R_(pore)) that models a resistance associated with the nanopore, whichcan change based on the presence a particular tag in the nanopore.

Voltage source V_(liq) 1320 is an alternating current (AC) voltagesource. Counter electrode 1316 is immersed in the bulk liquid 1318, andan AC non-Faradaic mode is utilized to modulate a square wave voltageV_(liq) and apply it to the bulk liquid in contact with the lipidmembranes/bilayers in the measurement cells. In some embodiments,V_(liq) is a square wave with a magnitude of ±200-250 mV and a frequencybetween 25 and 100 Hz.

Pass device 1306 is a switch that can be used to connect or disconnectthe lipid membrane/bilayer and the electrodes from the measurementcircuitry 1300. The switch enables or disables a voltage stimulus thatcan be applied across the lipid membrane/bilayer in the cell. Beforelipids are deposited to the cell to form a lipid bilayer, the impedancebetween the two electrodes is very low because the well of the cell isnot sealed, and therefore switch 1306 is kept open to avoid ashort-circuit condition. Switch 1306 may be closed once lipid solventhas been deposited to the cell that seals the well of the cell.

Circuitry 1300 further includes an on-chip fabricated integratingcapacitor 1308 (n_(cap)). Integrating capacitor 1308 is pre-charged byusing a reset signal 1303 to close switch 1301, such that integratingcapacitor 1308 is connected to a voltage source V_(pre) 1305. In someembodiments, voltage source V_(pre) 1305 provides a constant positivevoltage with a magnitude of 900 mV. When switch 1301 is closed,integrating capacitor 1308 is pre-charged to the positive voltage levelof voltage source V_(pre) 1305.

After integrating capacitor 1308 is pre-charged, reset signal 1303 isused to open switch 1301 such that integrating capacitor 1308 isdisconnected from voltage source V_(pre) 1305. At this point, dependingon the level of V_(liq), the potential of counter electrode 1316 may beat a higher level than the potential of working electrode 1314, or viceversa. For example, during the positive phase of square wave V_(liq)(i.e., the dark period of the AC voltage source signal cycle), thepotential of counter electrode 1316 is at a higher level than thepotential of working electrode 1314. Similarly, during the negativephase of square wave V_(liq) (i.e., the bright period of the AC voltagesource signal cycle), the potential of counter electrode 1316 is at alower level than the potential of working electrode 1314. Due to thispotential difference, integrating capacitor 1308 may be charged duringthe bright period of the AC voltage source signal cycle and dischargedduring the dark period of the AC voltage source signal cycle.

Depending on the sampling rate of an analog-to-digital converter (ADC)1310, integrating capacitor 1308 charges or discharges for a fixedperiod of time, and then the voltage stored in integrating capacitor1308 may be read out by ADC 1310. After the sampling by ADC 1310,integrating capacitor 1308 is pre-charged again by using reset signal1303 to close switch 1301, such that integrating capacitor 1308 isconnected to voltage source V_(pre) 1305 again. In some embodiments, thesampling rate of ADC 1310 is between 1500 to 2000 Hz. In someembodiments, the sampling rate of ADC 1310 is up to 5 kHz. For example,with a sampling rate of 1 kHz, integrating capacitor 1308 charges ordischarges for a period of ˜1 ms, and then the voltage stored inintegrating capacitor 1308 is read out by ADC 1310. After the samplingby ADC 1310, integrating capacitor 1308 is pre-charged again by usingreset signal 1303 to close switch 1301 such that integrating capacitor1308 is connected to voltage source V_(pre) 1305 again. The steps ofpre-charging the integrating capacitor 1308, waiting a fixed period oftime for the integrating capacitor 1308 to charge or discharge, andsampling the voltage stored in integrating capacitor by ADC 1310 arethen repeated in cycles throughout a lipid bilayer measurement phase ofthe system.

A digital processor 1330 can analyze the ADC values, e.g., fornormalization. The digital processor can be implemented as hardware(e.g., in a GPU, FPGA, ASIC) or as a combination of hardware andsoftware. In some embodiments, digital processor 1330 can performfurther downstream processing.

Circuitry 1300 can be used to detect whether a lipid bilayer is formedin the cell by monitoring a delta voltage change, ΔV_(ADC), atintegrating capacitor 1308 (n_(cap)) in response to a delta voltagechange (ΔV_(liq)) applied to the bulk liquid in contact with the lipidmembrane/bilayer. During the lipid bilayer measurement phase, circuitry1300 can be modeled as a voltage divider with C_(bilayer) 1326,C_(double layer) 1324, and n_(cap) 1308 connected in series, and avoltage change tapped at an intermediate point of the voltage dividercan be read by ADC 1310 for determining whether a lipid bilayer has beenformed.

Further details regarding the measurement circuitry can be found in, forexample, U.S. patent application Ser. No. 14/577,511 entitled“Nanopore-Based Sequencing With Varying Voltage Stimulus,” U.S. patentapplication Ser. No. 14/971,667 entitled “Nanopore-Based Sequencing WithVarying Voltage Stimulus,” U.S. patent application Ser. No. 15/085,700entitled “Non-Destructive Bilayer Monitoring Using Measurement OfBilayer Response To Electrical Stimulus,” and U.S. patent applicationSer. No. 15/085,713 entitled “Electrical Enhancement Of BilayerFormation.”

III. Sequencing Operation

To perform sequencing, the value of the ADC (e.g., 1310) can be measuredwhile a nucleotide is being added to a nucleic acid. The tag of thenucleotide can be pushed into the nanopore by the applied electric fieldacross the nanopore, when the applied electric field is such thatV_(liq) is higher than V_(pre).

A. Threading

A threading event in the context of a sequencing operation is when atagged nucleotide is being attached to the DNA fragment, and the taggoes in and out of the well. This can happen multiple times during athreading event. When the tag is in the well, a lower ADC measurement ofthe current will occur.

During threading, some cycles (i.e., of AC cycle) will not have the tagin the well. The bright mode is the mode where a tag might be attractedinto the well. A dark mode is when the tag is pushed out of the well.Open channel is when there is no tag in the well, and so the current isthe highest (V=IR).

B. Bright and Dark Cycles

In some embodiments, an AC voltage is applied across the system, e.g.,at 80 Hz. An acquisition rate of ADC can be about 1900 Hz. Thus, therecan be about 23-24 data points (voltage measurements) taken per AC cycle(cycle of AC square wave). There are sets of points per AC cycle (i.e.,sequencing cycle), where each set of points corresponds to one cycle ofthe AC waveform. In a set for an AC cycle, there is a subset for whenV_(liq) is higher than V_(pre), which is called a bright mode (channel),as that is when the tag can be forced into the nanopore. Another setcorresponds to a dark mode (channel) when the tag is pushed out of thenanopore by the applied electric field.

C. Decay Within a Data Acquisition Cycle and Decrease in MeasuredVoltages Within a Cycle

For each set of data points, when the switch 1301 is opened, the voltageat n_(cap) will change in a decaying manner, as an increase to V_(liq)when V_(liq) is higher than V_(pre) or a decrease to V_(liq) whenV_(liq) is lower than V_(pre). The measured voltage can be at apredetermined time relative to when the switch 1301 opens. This voltagemight be expected to be about the same for each measurement, but this isnot the case when charge builds up as C_(double layer) 1324. As aresult, the voltage is shifted, thereby causing the measured value todecrease for each data point in a cycle. Thus, within a cycle the datapoint values will change somewhat from one data point to the next withina cycle to be closer to V_(pre). The ΔADC value from V_(pre) decreasesfrom point to point within a cycle. A time constant of the system can beabout 200-500 ms.

Accordingly, when the switch 1301 is opened and an ADC value ismeasured, each data point is a result of a decay for charging ordischarging back to V_(pre). When the switch is closed, the ADC value isdriven back to V_(pre). The decays may not be measured fully, as onlyone data point is to be measured during each decay cycle, although highrates of measurement may be used. The decay is governed by the value ofthe resistance of the bilayer, which can include a nanopore, which canin turn include a molecule (e.g., tagged nucleotides).

The switch operates at the time of data acquisition. The switch would beclosed for a relatively short time between two acquisitions of data. Theswitch would typically change right after measurement of the ADC. Theswitch allows multiple data points to be collected for each cycle.Otherwise, the value of the ADC would decay to V_(liq), and stay there.Such multiple measurements can allow higher resolution with a fixed ADC(e.g., 8-bit to 14-bit due to the greater number of measurements, whichmay be averaged).

The multiple measurements also provide kinetic information, e.g., theycan provide information about the molecule threads into the nanopore.The timing information allows for a determination of how long athreading event lasts. This can be used in helping to determine whethermultiple nucleotides were added to the DNA strand being sequenced.Having the switch further allows for a voltage to be applied across thenanopore for longer periods of time, as otherwise the tag can move outof the nanopore, which again relates to only obtaining one data point.

FIG. 14 shows example data points captured from a nanopore cell duringbright periods and dark periods of AC cycles. In FIG. 14, the change inthe data points is exaggerated for illustration purpose. The voltage(V_(PRE)) applied to the working electrode or the integrating capacitoris at a constant level, such as, for example, 900 mV. A voltage signal1410 (V_(LIQ)) applied to the counter electrode of the nanopore cells isan AC signal shown as a rectangular wave, where the duty cycle may beany suitable value, such as less than or equal to 50%, for example,about 40%.

During a bright period 1420, voltage signal 1410 (V_(LIQ)) applied tothe counter electrode is lower than the voltage V_(PRE) applied to theworking electrode, such that a tag may be forced into the barrel of thenanopore by the electric field caused by the different voltage levelsapplied at the working electrode and the counter electrode (e.g., due tothe charge on the tag and/or flow of the ions). When switch 1301 isopened, the voltage at a node before the ADC (e.g., at an integratingcapacitor) will decrease. After a voltage data point is captured (e.g.,after a specified time period), switch 1301 may be closed and thevoltage at the measurement node will increase back to V_(PRE) again. Theprocess can repeat to measure multiple voltage data points. In this way,multiple data points may be captured during the bright period.

As shown in FIG. 14, a first data point 1422 (also referred to as firstpoint delta (FPD)) in the bright period after a change in the sign ofthe V_(LIQ) signal may be lower than subsequent data points 1424. Thismay be because there is no tag in the nanopore (open channel), and thusit has a low resistance and a high discharge rate. In some instances,first data point 1422 may exceed the V_(LIQ) level as shown in FIG. 14.This may be caused by the capacitance of the bilayer coupling the signalto the on-chip capacitor. Data points 1424 may be captured after athreading event has occurred, i.e., a tag is forced into the barrel ofthe nanopore, where the resistance of the nanopore and thus the rate ofdischarging of the integrating capacitor depends on the particular typeof tag that is forced into the barrel of the nanopore. Data points 1424may decrease slightly for each measurement due to charge built up atC_(Double Layer) 1324, as mentioned below.

During a dark period 1430, voltage signal 1410 (V_(LIQ)) applied to thecounter electrode is higher than the voltage (V_(PRE)) applied to theworking electrode, such that any tag would be pushed out of the barrelof the nanopore. When switch 1301 is opened, the voltage at themeasurement node increases because the voltage level of voltage signal1410 (V_(LIQ)) is higher than V_(PRE). After a voltage data point iscaptured (e.g., after a specified time period), switch 1301 may beclosed and the voltage at the measurement node will decrease back toV_(PRE) again. The process can repeat to measure multiple voltage datapoints. Thus, multiple data points may be captured during the darkperiod, including a first point delta 1432 and subsequent data points1434. As described above, during the dark period, any nucleotide tag ispushed out of the nanopore, and thus minimal information about anynucleotide tag is obtained, besides for use in normalization. Therefore,the output voltage signals from the cells during the dark period mayhave little or no use.

FIG. 14 also shows that during bright period 1440, even though voltagesignal 1410 (V_(LIQ)) applied to the counter electrode is lower than thevoltage (V_(PRE)) applied to the working electrode, no threading eventoccurs (open-channel). Thus, the resistance of the nanopore is low, andthe rate of discharging of the integrating capacitor is high. As aresult, the captured data points, including a first data point 1442 andsubsequent data points 1444, show low voltage levels.

The voltage measured during a bright or dark period might be expected tobe about the same for each measurement of a constant resistance of thenanopore (e.g., made during a bright mode of a given AC cycle while onetag is in the nanopore), but this may not be the case when charge buildsup at double layer capacitor 1324 (C_(Double Layer)). This chargebuild-up can cause the time constant of the nanopore cell to becomelonger. As a result, the voltage level may be shifted, thereby causingthe measured value to decrease for each data point in a cycle. Thus,within a cycle, the data points may change somewhat from data point toanother data point, as shown in FIG. 14.

D. Determining Bases

As part of calibration, various checks can be made during creation ofthe sequencing cell. Once a cell is created, further calibration stepscan be performed, e.g., to identify sequencing cells that are performingas desired (e.g., one nanopore in the cell). Such calibration checks caninclude physical checks, voltage calibration, open channel calibration,and identification of wells with single nanopore.

Once the usable cells of a chip are identified, a production mode can berun to sequence nucleic acids, one for each usable cell. The ADC valuesmeasured during sequencing can be normalized to provide greateraccuracy. Normalization can account for offset effects, such as cycleshape and baseline shift. After normalization, embodiments can determineclusters of voltages for the threaded channels, and use the clusters todetermine cutoff voltages for discriminating between different bases.

Further details regarding the sequencing operation can be found in, forexample, U.S. patent application Ser. No. 14/577,511 entitled“Nanopore-Based Sequencing With Varying Voltage Stimulus,” U.S. patentapplication Ser. No. 14/971,667 entitled “Nanopore-Based Sequencing WithVarying Voltage Stimulus,” U.S. patent application Ser. No. 15/085,700entitled “Non-Destructive Bilayer Monitoring Using Measurement OfBilayer Response To Electrical Stimulus,” and U.S. patent applicationSer. No. 15/085,713 entitled “Electrical Enhancement Of BilayerFormation,” which are incorporated by reference in their entirety.

IV. Osmotic Imbalance Methods for Stabilizing Nanopores

As discussed above, the nanopores of each sequencing cell can permit thetransfer of ions into and out of the well of the sequencing cell. Whenthe bias of the working electrode is positive relative to the counterelectrode, negative ions (anions) can be conducted from the externalreservoir into the well and positive ions (cations) can be conductedfrom the well into the external reservoir. When the bias is negative,cations are conducted from the external reservoir into the well andanions are conducted from the well into the external reservoir. Proteinpores such as alpha hemolysin (aHL) are known to preferentially conducteither anions or cations and to have unequal conductivity under positiveand negative electrical bias. These ion flow properties can lead to anet influx from or efflux into the well. If there is a net flow of ionsout of the well as a result of the bias, water will diffuse through thelipid bilayer from the well into the external reservoir to balance theirrespective electrolyte concentrations. As the volume of fluid in thewell is reduced, a resulting strain on the lipid bilayer can cause theinserted nanopore to leave the bilayer. If there is a net flow of ionsinto the well as a result of the bias, water will diffuse into the well.As the volume of water inside the well increases, the strain on thelipid bilayer can cause additional protein pores to insert into thelipid bilayer. In either case, the net transfer of ions between the welland the external reservoir tends to make a change to the ratio of thewell osmolarity to the external reservoir osmolarity.

The nanopores inserted in the planar lipid bilayers (PLBs) have beenfound to leave the planar lipid bilayers after an extended period ofapplied either alternating current (AC) or direct current (DC) voltagebetween the counter electrode and the working electrode. When theapplied voltage is significantly reduced, the lifetime of a nanoporeinserted in a lipid bilayer is increased. However, a minimum voltagemust be applied for tags to thread the nanopore and to measure thepresence of the tags in the nanopore. The reduction in nanopore lifetimelimits the number of nucleotides in the tag that may be read by thenanopore, thereby reducing the efficiency of the nanopore basedsequencing chip.

A. Comparative Example of Ion Efflux from Well Causing NanoporeInstability

FIG. 6 (including FIGS. 6A, 6B, and 6C) illustrates an embodiment inwhich a voltage applied across a lipid bilayer for nucleic acidsequencing over time causes an osmotic imbalance between the electrolytesolution above and below the lipid bilayer, which pulls the lipidbilayer inward into the well and causes the nanopore to be released fromthe lipid bilayer.

FIG. 6A illustrates that initially at time t₁, a nanopore 606 isinserted into a planar lipid bilayer 604 spanning across a well 602 in acell of the nanopore based sequencing chip. The planar lipid bilayer 604separates the well from a reservoir 608 external to the well. Initiallyat time t₁, the osmolarity of the salt/electrolyte solution within thewell, [E_(w)], is the same as the osmolarity of the bulk electrolytesolution in the external reservoir, [E_(R)]. Osmolarity, also known asosmotic concentration, is a measure of solute concentration.

FIG. 6B illustrates that later at time t₂, as a voltage is appliedacross the lipid bilayer, an osmotic imbalance between the electrolytesolution above and below the lipid bilayer occurs. In this example, theosmotic imbalance is caused by a net efflux of ions out of the well,which causes water to diffuse out of the well through the lipid bilayerdue to osmosis, as will be described in greater detail next.

When a voltage is applied across the lipid bilayer and the nanopore, thenanopore conducts both positive ions (cations) and negative ions(anions) into and out of the well. For example, when an electrolytesolution of potassium chloride (KCl) fills the well and the externalreservoir, positive K⁺ ions and negative Cl⁻ ions flow into and out ofthe well. In particular, when the bias of the working electrode ispositive relative to the counter electrode, negative ions are conductedfrom the reservoir into the well and positive ions are conducted fromthe well into the reservoir. Conversely, when the bias of the workingelectrode is negative relative to the reference electrode, positive ionsare conducted from the reservoir into the well and negative ions areconducted from the well into the reservoir.

Some nanopores, such as alpha hemolysin (aHL), preferentially conducteither anions or cations and have unequal conductivity under positiveand negative electrical bias. Because of these properties, a net influxof ions into or a net efflux of ions out of the well can be observed. Ifthere is a net efflux of ions flowing out of the well, then theosmolarity of the salt/electrolyte solution within the well ([E_(w)])decreases and transiently falls below the osmolarity of the bulkelectrolyte solution in the external reservoir ([E_(R)]) (i.e.,[E_(w)]<[E_(R)]), creating an osmolarity gradient across the lipidbilayer. To equilibrate the electrolyte osmolarity in the well and theexternal reservoir, water diffuses through the planar lipid bilayer fromthe well into the external reservoir, as shown in FIG. 6B.

FIG. 6C illustrates that later at time t₃, a net flow of water acrossthe lipid bilayer places a strain on the lipid bilayer, causing thelipid bilayer to rupture or change its shape to a point that it fails towork or causing the nanopore to leave the lipid bilayer. In thisexample, the net efflux of ions out of the well causes water to diffuseout of the well, and the resulting loss of water from the well pulls thelipid bilayer inward, causing a change in the shape of the lipid bilayerto a point at which the lipid bilayer fails to work or causing thenanopore to leave the lipid bilayer.

B. Comparative Example of Ion Influx into Well Causing NanoporeInstability

FIG. 7 (including FIGS. 7A, 7B, and 7C) illustrates an embodiment inwhich a voltage applied across a lipid bilayer for nucleic acidsequencing over time causes an osmotic imbalance between the electrolytesolution above and below the lipid bilayer, which pushes the lipidbilayer outward from the well.

FIG. 7A illustrates that initially at time t₁, a nanopore 706 isinserted into a planar lipid bilayer 704 spanning across a well 702 in acell of the nanopore based sequencing chip. The planar lipid bilayer 704separates the well from a reservoir 708 external to the well. Initiallyat time t₁, the osmolarity of the salt/electrolyte solution within thewell ([E_(w)]) is the same as the osmolarity of the bulk electrolytesolution in the external reservoir ([E_(R)]).

FIG. 7B illustrates that later at time t₂, as a voltage is appliedacross the lipid bilayer, an osmotic imbalance between the electrolytesolution above and below the lipid bilayer occurs. In this example,there is a net influx of ions flowing into the well, and the osmolarityof salt/electrolyte solution within the well ([E_(w)]) increases andtransiently rises above the osmolarity of the bulk electrolyte solutionin the external reservoir ([E_(R)]) (i.e., [E_(w)]>[E_(R)]), creating anosmolarity gradient across the lipid bilayer. To equilibrate theelectrolyte osmolarity in the well and the external reservoir, waterdiffuses through the planar lipid bilayer from the external reservoirinto the well, as shown in FIG. 7B.

FIG. 7C illustrates that later at time t₃, a net flow of water acrossthe lipid bilayer and the resulting increase in volume of water in thewell pushes the lipid bilayer outward, causing a change in the shape ofthe lipid bilayer to a point that the lipid bilayer fails to work. Asthe volume of water inside the well increases, the strain on the planarlipid bilayer may also cause additional nanopores to insert into thelipid bilayer.

C. Counterbalancing Osmotic Imbalances

FIG. 8 illustrates an embodiment of a process 800 for an improvedtechnique of extending the lifetime of a nanopore inserted in a lipidbilayer in a cell of a nanopore based sequencing chip for analyzingmolecules. The improved technique applies an electrolyte flow over theplanar lipid bilayer, wherein the electrolyte flow has a differentosmolarity (either a lower or higher osmotic concentration, depending onthe net direction of ion transfer through the nanopore) than theosmolarity of the electrolyte below the planar lipid bilayer. In oneembodiment, the electrolyte flow over the lipid bilayer is applied priorto or during the application of voltage across the lipid bilayer fornucleic acid sequencing. The disclosed technique has many advantages,including increasing the nanopore lifetime and increasing the efficiencyand yield of the nanopore based sequencing chip. It is also appreciatedthat the disclosed technique can be applied to other semi-permeablemembranes (e.g., instead of the lipid bilayer) that permit thetransmembrane flow of water but have limited to no permeability to theflow of ions. In some embodiments, the nanopore based sequencing chipused for the process of FIG. 8 includes a plurality of cells 100 ofFIG. 1. In some embodiments, the nanopore based sequencing chip used forthe process of FIG. 8 includes a plurality of cells 500 of FIG. 5.

In step 802 of process 800, a lipid bilayer is formed in each of thecells of the sequencing chip. The lipid bilayer divides the well of eachof the cells from a reservoir external to the well (i.e., a firstreservoir). In step 804 of process 800, after a lipid bilayer is formedin a cell a nanopore is inserted into the lipid bilayer. In someembodiments, and as shown in FIG. 8, the nanopore is inserted into thelipid bilayer before an electrolyte solution is flowed to the externalreservoir in step 806. In some embodiments, the nanopore is insertedinto the lipid bilayer after the electrolyte solution is flowed to theexternal reservoir. Different techniques can be used to insert nanoporesin the cells of the nanopore based sequencing chip. In some embodiments,a solution containing a nanopore forming protein or polypeptide (e.g.,α-hemolysin) is flowed through the cells of the nanopore basedsequencing chip via the flow chamber such that the solution is flowedabove the lipid bilayers. In some embodiments, an agitation orelectrical stimulus (e.g., ˜100 mV to 1.0 V for 50 ms to 1 s) is appliedacross the lipid bilayer membrane, causing a disruption in the lipidbilayer and initiating the insertion of an α-hemolysin nanopore into thelipid bilayer.

In step 806 of process 800, a salt/electrolyte buffer solution is flowedthrough the cells of the nanopore based sequencing chip via the flowchamber. The concentration or osmolarity of the salt electrolyte buffersolution is selected, as described in more detail below, so as tointroduce a particular initial osmotic imbalance between the electrolytesolutions above and below the lipid bilayer. This initial osmoticimbalance is characterized by a change to the ratio of the externalreservoir (i.e., first reservoir) osmolarity and the well (i.e., secondreservoir) osmolarity. In other words, the flowing of the electrolytesolution to the external reservoir tends to make a change to the ratioof the external reservoir osmolarity to the well osmolarity (i.e., aninitial osmotic imbalance). The initial osmotic imbalance tends to besubstantially canceled out, or counterbalanced, by an opposite osmoticimbalance that is caused by a net transfer of ions through the nanoporeduring, for example, a subsequent nucleic acid sequencing operation. Inthe absence of the initial osmotic imbalance, such a net ion transfercan create a structural strain on the lipid bilayer for a time periodsufficient to negatively affect the integrity of a nanopore insertedtherein. In contrast, with the initial osmotic imbalance acting tosubstantially counterbalance the effects of a later net ion transfer,such structural strains can be reduced, eliminated, or made moretransient, thereby improving the integrity and effective lifetime of theinserted nanopore.

In some embodiments, the distortion of the lipid bilayer as a result ofthe electrolyte solution flow can have a similar magnitude to thedistortion resulting from the application of voltages during sequencingoperations, e.g., when a DC voltage is applied. However, the length oftime that the distortion driven by the electrolyte solution flow existscan be significantly smaller than the time scale of the sequencingoperations. For example, the distortion due to the electrolyte solutionflow may exist for only the short time that it takes to establish theosmolarity imbalance, until sequencing starts. As a result, the moretransient nature of the distortions causes less structural strain on thelipid bilayers, increasing their stability and integrity, and decreasingthe chances of nanopore loss.

In other embodiments, the distortion of the lipid bilayer as a result ofthe electrolyte solution flow can have a smaller magnitude than thedistortion resulting from the application of voltages during sequencingoperations, e.g., when AC voltage is applied. For example, the initialdistortion (before sequencing) can be slightly outward, at a magnitudeof half of the distortion caused by the positive part of the AC signal,but in the opposite direction of distortion. Then, once the AC signalstarts (e.g., positive part first), the lipid bilayer can becomedistorted inward, at half of the magnitude caused by the voltage. Then,the negative part of the AC signal can cause the lipid bilayer todistort outward again (due to the different flux of anions/cations),thereby causing the distortion to be outward again to reach the initialdistortion. In this manner, the distortion is never at the fullmagnitude that would result from the voltage being applied to a cellthat had osmolarity balance. Accordingly, in some embodiments, thedistortion of the lipid bilayer can match the distortion due to anapplied voltage or be less.

For a type of nanopore that is known to produce a net efflux of ionsfrom the well in response to an applied voltage, the osmolarity of thesalt/electrolyte solution within the well ([E_(w)]) can be expected todecrease. As a result, the well osmolarity can fall below the osmolarityof the bulk electrolyte solution in the external reservoir ([E_(R)]) Inother words, for ion effluxing nanopores, [E_(R)]/[E_(w)] can increaseand can be >1. To equilibrate the electrolyte osmolarity in the well andthe external reservoir, water can be expected to diffuse through theplanar lipid bilayer from the well into the external reservoir, as shownpreviously in FIG. 6B.

To counterbalance the expected increasing [E_(R)]/[E_(w)] osmolarityratio, the concentration or osmolarity of the salt electrolyte buffersolutionof step 806 of process 800 is selected as to decrease the[E_(R)]/[E_(w)] ratio, changing the ratio in an opposite direction. Thiscan have the effect of driving excess water into the well. For example,the salt electrolyte buffer solution that is flowed through the cells ofthe nanopore based sequencing chip via the flow chamber at step 806 canhave a lower concentration (e.g., 300 mM) than the electrolyte solutionthat is present in the well (e.g., 340 mM). In response to the lowerelectrolyte concentration in the solution flowing in the externalreservoir (i.e., on the cis side of the planar lipid bilayer), waterdiffuses across the planar lipid bilayer from the reservoir into thewell in order to equalize the concentration on the cis and trans sidesof the lipid bilayer. This equalization can take place almostinstantaneously since water molecules can readily flow through theplanar lipid bilayer. The concentrations on both sides of the planarlipid bilayer can equalize to that of the cis side (e.g., 300 mM) sincethe volume of the external reservoir is significantly greater than thatof the trans side (the well). This can effectively increase the volumeof water under the planar lipid bilayer in the well, causing the planarlipid bilayer to bow upwards.

For a type of nanopore that is known to produce a net influx of ionsinto the well in response to an applied voltage, the osmolarity of thesalt/electrolyte solution within the well ([E_(w)]) is expected toincrease and transiently rise above the osmolarity of the bulkelectrolyte solution in the external reservoir ([E_(R)]) (i.e.,[E_(R)]/[E_(w)] is decreasing and is <1). To equilibrate the electrolyteosmolarity in the well and the external reservoir, water is expected todiffuse through the planar lipid bilayer from the external reservoirinto the well. To counterbalance the expected decreasing [E_(R)]/[E_(w)]osmolarity ratio, the concentration or osmolarity of the saltelectrolyte buffer solution is determined by process 800 so as toincrease the [E_(R)]/[E_(w)] ratio, which will in turn force excesswater out of the well. For example, the salt electrolyte buffer solutionthat is flowed through the cells of the nanopore based sequencing chipvia the flow chamber in step 806 has a higher concentration (e.g., 340mM) than the electrolyte solution that is present in the well (e.g., 300mM). In response to the higher concentration electrolyte solutionflowing in the external reservoir (i.e., on the cis side of the planarlipid bilayer), water diffuses across the planar lipid bilayer from thewell into the reservoir in order to equalize the concentration on thecis and trans sides of the lipid bilayer. This equalization takes placealmost instantaneously since the water molecules readily flow throughthe planar lipid bilayer. The concentrations on both sides of the planarlipid bilayer equalize to that of the cis side (e.g., 340 mM) since thevolume of the external reservoir is significantly greater than that ofthe trans side (the well). This effectively decreases the volume ofwater under the planar lipid bilayer in the well, causing the planarlipid bilayer to bow downwards.

The concentration of the electrolyte solution in step 806 of process 800can be selected based on different factors. The concentration differencebetween the initial flow and the concentration of the electrolytesolution in the well can, for example, be selected or optimized tomaximize nanopore lifetime, limit rapid insertions of nanopores in theplanar lipid bilayers, or to avoid rupture of the planar lipid bilayers.In some embodiments, the concentration of the electrolyte solution isselected such that the first change to the osmolarity ratio (caused bythe electrolyte solution flow) substantially counterbalances the secondchange to the osmolarity ratio (caused by the voltage application). Insome embodiments, the concentration of the electrolyte solution isselected such that the first change to the osmolarity at least partiallycounterbalances the second change to the osmolarity ratio. It isappreciated that any first change to the osmolarity ratio that is in anopposite direction to the second change to the osmolarity ratio will besufficient to at least partially reduce the resulting distortion of thelipid bilayer.

In step 810 of process 800, it is determined whether the flowing of theelectrolyte solution (in step 806) should be repeated. Differentcriteria can be used in this step. In some embodiments, step 806 isperformed a predetermined number of times. The concentration ofelectrolytes in the electrolyte solution can be identical, similar, ordifferent for each iteration of step 806. Lower or higher concentrationsof electrolytes can be applied for one or multiple additional cycles.For example, in the case in which the nanopores are known to produce anet efflux of ions from the well, each time step 806 is repeated theconcentration of the salt electrolyte solution can be progressivelylowered from an initial electrolyte concentration or solution osmolarity(i.e., the conditions for a first iteration of step 806) to a finalelectrolyte concentration or solution osmolarity (i.e., the conditionsfor a last iteration of step 806), until the [E_(R)]/[E_(w)] ratio isdecreased to a predetermined target ratio. This ratio can be estimatedby using osmolarity measurements of the external reservoir fluid exitingthe system. In the case in which the nanopores are known to produce anet influx of ions into the well, each time step 806 is repeated theconcentration of the salt electrolyte solution can be progressivelyincreased from an initial electrolyte concentration or solutionosmolarity to a final electrolyte concentration or solution osmolarityuntil the [E_(R)]/[E_(w)] ratio is increased to a predetermined targetratio. If the flowing of the electrolyte solution (in step 806) isrepeated, process 800 proceeds to step 806 from step 810; otherwise,process 800 proceeds to step 812.

In FIG. 8, the osmotic imbalance of step 806 of process 800 is shown tobe introduced after step 804. In this case, the nanopore is insertedinto the lipid bilayer prior to the flowing of the electrolyte solutionto adjust an osmotic imbalance. As discussed above, in otherembodiments, steps 806 and step 810 can be performed during or beforestep 804. In these cases, the nanopore is inserted during or after thecreation of an osmotic imbalance.

In step 812 of process 800, nucleic acid sequencing is performed asdescribed above. The sequencing operations can include the applying of avoltage across the lipid bilayer, wherein the voltage causes a nettransfer of ions between the external reservoir and the well via thenanopore. The ion transfer can make a second change to the ratio of theexternal reservoir (i.e., first reservoir) osmolarity to the well (i.e.,second reservoir) osmolarity. Because of the first change to theosmolarity ratio caused by the electrolyte solution flow of step 806,this second change to the osmolarity ratio is substantiallycounterbalanced, and the lipid bilayer can return to its originalconformation, without significant bowing or distortion. Process 800 canthen be repeated for other cycles of introducing an osmotic imbalancethrough electrolyte flow, and counterbalancing through the applicationof a voltage during sequencing. In some embodiments, process 800 isoperated concurrently with sequencing operations after sequencing hasbegun. Process 800 can be operated continuously, semi-continuously, ordiscretely as needed to enhance the effective lifetime or efficiency ofthe sequencing chip and the lipid bilayers and nanopores therein.

V. Improved Flow Chamber

Process 800 includes steps (e.g., steps 802, 804, and 806) in whichdifferent types of fluids (e.g., liquids or gases) are flowed throughthe cells of the nanopore based sequencing chip via a flow chamber.Multiple fluids with significantly different properties (e.g.,compressibility, hydrophobicity, and viscosity) can be flowed over anarray of sensor cells (e.g., like cell 100 of FIG. 1 or cell 500 of FIG.5) on the surface of the nanopore based sequencing chip during this andother processes. The efficiencies of these processes can be improved byexposing each of the sensor cells (also called “sensors”) in the arrayto the fluids in a consistent manner. For example, each of the differenttypes of fluids can be flowed over the nanopore based sequencing chipsuch that the fluid evenly coats and contacts each of the cells'surfaces before the fluids are delivered out of the chip. As describedabove, a nanopore based sequencing chip can incorporate a large numberof sensor cells configured as an array. As the nanopore based sequencingchip is scaled to include more and more cells, achieving an even flow ofthe different types of fluids across the cells of the chip becomes morechallenging.

In some embodiments, the nanopore based sequencing system that performsprocess 800 of FIG. 8 includes an improved flow chamber having aserpentine fluid flow channel that directs the fluids to traverse overdifferent sensors of the chip along the length of the channel. The flowchannel can, for example, be used to contain the bulk electrolyte 114 inFIG. 1 or the bulk electrolyte 508 in FIG. 5. The flow channel can beused to form external reservoir 522 in FIG. 5, external reservoir 608 inFIG. 6, or external reservoir 708 in FIG. 7.

FIG. 9 illustrates the top view of a nanopore based sequencing system900 with an improved flow chamber enclosing a silicon chip. A serpentineor winding flow channel 908 directs fluids contained within to flowdirectly above a series of sensor banks 906, e.g., a row or a column ofsensors, which can include several thousands of sensor cells, until allof the sensor banks on the chip surface have been traversed at leastonce. The serpentine configuration of the flow channel allows fluid to:enter the channel through inlet 902, travel along a column or row ofsensor banks, repeatedly loop back to travel along an adjacent column orrow, and then exit the channel through outlet 904. Each of the sensorbanks can include an array of sequencing cells. In some embodiments,each sensor bank includes several thousand sequencing cells.

The type of fluid, the concentration of the fluid, or the flow speed ofsequencing system 900 can be selected by a fluidic system controlled bya processor. Inlet 902 can be a tube or a needle. For example, the tubeor needle can have a diameter of one millimeter. This is in contrast toalternative embodiments without a serpentine channel, in which theliquid or gas is instead inserted directly into the entire width of theflow chamber. The serpentine channel 908 can be formed by stackingtogether a top plate and a gasket with dividers 910 that divide thechamber into the serpentine channel to form a flow cell, and thenmounting the flow cell on top of the chip. Once the liquid or gas flowsthrough the serpentine channel 908, the liquid or gas can be directed upthrough outlet 904 and out of system 900.

System 900 allows the fluids to flow more evenly on top of all thesensors on the chip surface. The channel width can be configured to benarrow enough such that capillary action has an effect. Moreparticularly, the surface tension (which is caused by cohesion withinthe fluid) and adhesive forces between the fluid and the enclosingsurfaces can act to hold the fluid together, thereby preventing thefluid or the air bubbles from breaking up and creating dead zones. Forexample, the channel can have a width of 1 millimeter or less. Thenarrow channel can enable controlled flow of the fluids and minimize theamount of remnants from a previous flow of fluids or gases.

VI. Forming of Lipid Bilayer

Different techniques can be used to form the lipid bilayers in the cellsof the nanopore based sequencing chip, e.g., as is done in step 802 ofprocess 800. For illustration purposes only, one exemplary process 1000for forming the lipid bilayers is shown in FIG. 10.

In step 1002 of process 1000, a salt/electrolyte buffer solution isflowed through the cells of the nanopore based sequencing chip via theflow chamber to substantially fill the wells in the cells with the saltbuffer solution. As further described herein, the salt buffer solutioncan include at least one of the following osmolytes: lithium chloride(LiCl), sodium chloride (NaCl), potassium chloride (KCl), lithiumglutamate, sodium glutamate, potassium glutamate, lithium acetate,sodium acetate, potassium acetate, calcium chloride (CaCl₂), strontiumchloride (SrCl₂), manganese chloride (MnCl₂), and magnesium chloride(MgCl₂).

In one aspect, the present invention provides a concentration of thesolution (e.g., salt solution or salt buffer solution) in the well(e.g., 506 in FIG. 5) that is higher than the concentration of solutionin the external reservoir (e.g., reservoir 522 in FIG. 5). In anotherembodiment, the external reservoir is a first reservoir characterized bya first reservoir osmolarity and the well is a second reservoircharacterized by a second reservoir osmolarity. In one embodiment, theconcentration of solution in the external reservoir is between about 10nM and 3 M. In another embodiment, the concentration of solution in theexternal reservoir is about 10 mM, about 20 mM, about 30 mM, about 40mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM,about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM,about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM,about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM,about 250 mM, about 260 mM, about 270 mM, about 280 mM, about 290 mM,about 300 mM, 305 mM, about 310 mM, about 315 mM, about 320 mM, about325 mM, about 330 mM, about 335 mM, about 340 mM, about 345 mM, about350 mM, about 355 mM, about 360 mM, about 365 mM, about 370 mM, about375 mM, about 380 mM, about 385 mM, about 390 mM, about 395 mM, about400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM, about900 mM, about 950 mM, about 1 M, about 1.25 M, about 1.5 M, about 1.75M, about 2 M, about 2.25 M, about 2.5 M, about 2.75 M, or about 3 M. Inanother embodiment, the concentration of solution in the well is about305 mM, about 310 mM, about 315 mM, about 320 mM, about 325 mM, about330 mM, about 335 mM, about 340 mM, about 345 mM, about 350 mM, about355 mM, about 360 mM, about 365 mM, about 370 mM, about 375 mM, about380 mM, about 385 mM, about 390 mM, about 395 mM, about 400 mM, about450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about700 mM, about 750 mM, about 800 mM, about 850 mM, about 900 mM, about950 mM, or about 1 M. In one additional embodiment, the concentration ofsolution in the external reservoir is about 300 mM and the concentrationof solution in the well is selected from the group consisting of about310 mM, about 320 mM, about 330 mM, about 340 mM, about 350 mM, about360 mM, about 370 mM, about 380 mM, about 390 mM, or about 400 mM. Inother embodiments, the concentration of solutions is selected from thegroup consisting of (i) 300 mM in the external reservoir and 310 mM inthe well, (ii) 300 mM in the external reservoir and 320 mM in the well,(iii) 300 mM in the external reservoir and 330 mM in the well, (iv) 300mM in the external reservoir and 340 mM in the well, (v) 300 mM in theexternal reservoir and 350 mM in the well, (vi) 300 mM in the externalreservoir and 360 mM in the well, (vii) 300 mM in the external reservoirand 370 mM in the well, (viii) 300 mM in the external reservoir and 380mM in the well, (ix) 300 mM in the external reservoir and 390 mM in thewell, and (x) 300 mM in the external reservoir and 400 mM in the well.

In step 1004 of process 1000, a lipid and solvent mixture is flowedthrough the cells of the nanopore based sequencing chip via the flowchamber. In some embodiments, the lipid and solvent mixture includeslipid molecules such as diphytanoylphosphatidylcholine or1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), and1,2-di-O-phytanyl-sn-glycero-3-phosphocholine (DOPhPC). In someembodiments, the lipid and solvent mixture includes decane or tridecane.When the lipid and solvent mixture is first deposited into the cells toform the lipid bilayers, some of the cells can have lipid bilayersspontaneously formed, but some of the cells can merely have a thicklipid membrane (with multiple layers of lipid molecules and solventcombined together) spanning across each of the wells of the cells.

In step 1006 of process 1000, a salt/electrolyte buffer solution isflowed through the cells of the nanopore based sequencing chip via theflow chamber to substantially fill the external reservoir with the saltbuffer solution.

In step 1008, in order to increase the yield of the nanopore basedsequencing chip (i.e., the percentage of cells in the nanopore basedsequencing chip with properly formed lipid bilayers and nanopores), oneor more types of lipid bilayer initiating stimuli can be applied to thenanopore based sequencing chip to facilitate the formation of lipidbilayers in additional cells. One or more types of lipid bilayerinitiating stimuli can be applied simultaneously, or in differentorders, during a lipid bilayer initiating stimulus phase (step 1008),which can be repeated (determined by step 1010) a plurality of times.

A lipid bilayer initiating stimulus facilitates the creation of a smalllipid bilayer on a thick lipid membrane. Once a small transient lipidbilayer on a thick lipid membrane is formed, the application ofadditional lipid bilayer initiating stimuli acts as a positive feedbackto continue to enlarge the surface area of the lipid bilayer. As aresult, the time required to form lipid bilayers in the cells of thenanopore based sequencing chip can be significantly reduced. One type oflipid bilayer initiating stimulus is a mechanical stimulus, such as avibration stimulus. Another type of lipid bilayer initiating stimulus isan electrical stimulus. Those of ordinary skill in the art willappreciate that other types of stimulus may be suitable for use with thepresent invention. Another type of lipid bilayer initiating stimulus isa physical stimulus. For example, flowing a salt/electrolyte buffersolution through the cells of the nanopore based sequencing chip via aflow chamber facilitates the formation of a lipid bilayer over each ofthe cells. The salt buffer solution flowed over the cells facilitatesthe removal of any excess lipid solvent such that the thick lipidmembranes can be thinned out and transitioned into lipid bilayers moreefficiently.

VII. Benefits of Counterbalancing Osmotic Imbalances

The provided counterbalancing osmotic imbalance methods and systemsoffer several benefits that can include increased longevity of nanoporesand sequencing cells, greater percentages of functional cells withinsequencing arrays, and higher efficiencies of instruments. Thesebenefits arise from the ability of the osmotic imbalance to counteractthe potentially destructive effects of ion and water flow between thetwo sides of the lipid bilayer, which in the absence of counterbalancingcan add conformational stress to the bilayer and cause rupture ornanopore loss as described further in the illustrations below

A. Illustration of Prevention of Ejection of Pore

FIG. 11A illustrates that by flowing over a lipid bilayer at time t₁ alower concentration of electrolyte solution than is initially present inthe well while the planar lipid bilayer is in place between the well andthe external reservoir, excess water is forced into the well, causingthe planar lipid bilayer to bow upwards. As discussed above, since (1)water can diffuse across the planar lipid bilayers, (2) ions can passthrough the nanopores, and (3) the salt electrolyte buffer solution thatis flowed through the cells can introduce different osmolytes into theexternal reservoir over time, both the volume and the osmolyte contentof the liquid held in the external reservoir and the wells can changeover time. It is recognized that the external reservoir can becharacterized by a first reservoir osmolarity, which is the osmolarityof the liquid contained in the external reservoir at a specific time. Awell in a cell can also be characterized by a second reservoirosmolarity, which is the osmolarity of the liquid contained in the welland confined by the lipid bilayer at a specific time.

FIG. 11B illustrates that the volume of water forced into the well attime t₁ (FIG. 11A) due to the initial flow of lower concentrationelectrolyte acts to substantially counterbalance a volume of waterremoved from the well at a later time t₂. The removal of water from thewell at time t₂ can be, for example, as a result of voltage appliedduring sequencing operation described above. Because the methods andsystems described herein create an osmotic imbalance that substantiallycounterbalances the effects of such a voltage application, the“pre-bowed” well is capable of withstanding the removal of a largervolume of water before the planar lipid bilayer ruptures or the nanoporeinserted therein is forced to exit.

FIG. 11C illustrates that in a comparative method lacking theapplication of a counteracting osmotic imbalance, the volume of waterremoved from the well at a later time t₂ can be sufficiently large so asto disrupt the lipid bilayer or eject its inserted nanopore. In thefigure it can be seen that the inward bowing of the bilayer releases thenanopore, ending the effectiveness of the cell for use in sequencingoperations. This is in contrast to the illustration of FIG. 11B, inwhich the outward and inward bowing caused by the electrolyte flow andthe voltage application, respectively, substantially cancel one anotherout, leaving the nanopore and lipid bilayer intact.

B. Example of Increasing Effective Nanopore Lifetime withCounterbalancing Osmatic Imbalances

FIGS. 12A, 12B, and 12C illustrate that using process 800, the averagelifetime of a nanopore in a cell is significantly increased. For eachchart, the y-axis represents the number of nanopores, and the x-axisrepresents the lifetime of the nanopores in units of 100 seconds asobserved during sequencing operations. In FIG. 12A, the concentrationsof both the electrolyte solution that is flowed through the cells of thesequencing chip via the flow chamber, and the electrolyte solution inthe well are 300 mM. As shown in the graph, the average lifetime of thenanopores is around 1500 seconds. This corresponds with a comparativemethod (as in, for example, FIG. 11C), in which no osmotic imbalance isapplied prior to sequencing, and nanopore loss can be occurring throughbilayer distortion brought about by voltage applications.

In the graph of FIG. 12B, the concentration of the electrolyte solutionthat is flowed through the cells of the nanopore based sequencing chipvia the flow chamber is 300 mM, and the concentration of the electrolytesolution in the well is 340 mM. This corresponds with a method in whichan osmotic imbalance is applied, water will be driven into the wells toequilibrate osmolyte concentrations, and the bilayer will “pre-bow”outward and substantially counterbalance bilayer distortion broughtabout by voltage applications (as in, for example, FIGS. 11A and 11B).The average lifetime of the nanopores in this case is increased toaround 3200 seconds.

In the graph of FIG. 12C, the concentration of the electrolyte solutionthat is flowed through the cells of the nanopore based sequencing chipvia the flow chamber is 300 mM and the concentration of the electrolytesolution in the well is 360 mM. The average lifetime of the nanopores isfurther increased to around 3800 seconds. This also corresponds with amethod in similar to that producing the results of the FIG. 12B graph,but with the creation of a larger osmotic imbalance that furtherimproves the nanopore effective lifetime. Such experimentation can becontinued in an iterative fashion to empirically derive or optimizeelectrolyte concentrations that are effective in improving nanoporestability and sequencing cell robustness.

VIII. Computer System

Any of the computer systems mentioned herein can utilize any suitablenumber of subsystems. Examples of such subsystems are shown in FIG. 15in computer system 10. In some embodiments, a computer system includes asingle computer apparatus, where the subsystems can be the components ofthe computer apparatus. In other embodiments, a computer system caninclude multiple computer apparatuses, each being a subsystem, withinternal components. A computer system can include desktop and laptopcomputers, tablets, mobile phones and other mobile devices.

The subsystems shown in FIG. 15 are interconnected via a system bus 75.Additional subsystems such as a printer 74, keyboard 78, storagedevice(s) 79, monitor 76, which is coupled to display adapter 82, andothers are shown. Peripherals and input/output (I/O) devices, whichcouple to I/O controller 71, can be connected to the computer system byany number of means known in the art such as input/output (I/O) port 77(e.g., USB, FIREWIRE®). For example, I/O port 77 or external interface81 (e.g. Ethernet, Wi-Fi, etc.) can be used to connect computer system10 to a wide area network such as the Internet, a mouse input device, ora scanner. The interconnection via system bus 75 allows the centralprocessor 73 to communicate with each subsystem and to control theexecution of a plurality of instructions from system memory 72 or thestorage device(s) 79 (e.g., a fixed disk, such as a hard drive, oroptical disk), as well as the exchange of information betweensubsystems. The system memory 72 and/or the storage device(s) 79 mayembody a computer readable medium. Another subsystem is a datacollection device 85, such as a camera, microphone, accelerometer, andthe like. Any of the data mentioned herein can be output from onecomponent to another component and can be output to the user.

A computer system can include a plurality of the same components orsubsystems, e.g., connected together by external interface 81, by aninternal interface, or via removable storage devices that can beconnected and removed from one component to another component. In someembodiments, computer systems, subsystem, or apparatuses can communicateover a network. In such instances, one computer can be considered aclient and another computer a server, where each can be part of a samecomputer system. A client and a server can each include multiplesystems, subsystems, or components.

Aspects of embodiments can be implemented in the form of control logicusing hardware circuitry (e.g. an application specific integratedcircuit or field programmable gate array) and/or using computer softwarewith a generally programmable processor in a modular or integratedmanner. As used herein, a processor can include a single-core processor,multi-core processor on a same integrated chip, or multiple processingunits on a single circuit board or networked, as well as dedicatedhardware. Based on the disclosure and teachings provided herein, aperson of ordinary skill in the art will know and appreciate other waysand/or methods to implement embodiments of the present invention usinghardware and a combination of hardware and software.

Any of the software components or functions described in thisapplication may be implemented as software code to be executed by aprocessor using any suitable computer language such as, for example,Java, C, C++, C#, Objective-C, Swift, or scripting language such as Perlor Python using, for example, conventional or object-orientedtechniques. The software code may be stored as a series of instructionsor commands on a computer readable medium for storage and/ortransmission. A suitable non-transitory computer readable medium caninclude random access memory (RAM), a read only memory (ROM), a magneticmedium such as a hard-drive or a floppy disk, or an optical medium suchas a compact disk (CD) or DVD (digital versatile disk), flash memory,and the like. The computer readable medium may be any combination ofsuch storage or transmission devices.

Such programs may also be encoded and transmitted using carrier signalsadapted for transmission via wired, optical, and/or wireless networksconforming to a variety of protocols, including the Internet. As such, acomputer readable medium may be created using a data signal encoded withsuch programs. Computer readable media encoded with the program code maybe packaged with a compatible device or provided separately from otherdevices (e.g., via Internet download). Any such computer readable mediummay reside on or within a single computer product (e.g. a hard drive, aCD, or an entire computer system), and may be present on or withindifferent computer products within a system or network. A computersystem may include a monitor, printer, or other suitable display forproviding any of the results mentioned herein to a user.

Any of the methods described herein may be totally or partiallyperformed with a computer system including one or more processors, whichcan be configured to perform the steps. Thus, embodiments can bedirected to computer systems configured to perform the steps of any ofthe methods described herein, potentially with different componentsperforming a respective steps or a respective group of steps. Althoughpresented as numbered steps, steps of methods herein can be performed ata same time or in a different order. Additionally, portions of thesesteps may be used with portions of other steps from other methods. Also,all or portions of a step may be optional. Additionally, any of thesteps of any of the methods can be performed with modules, units,circuits, or other means for performing these steps.

The specific details of particular embodiments may be combined in anysuitable manner without departing from the spirit and scope ofembodiments of the invention. However, other embodiments of theinvention may be directed to specific embodiments relating to eachindividual aspect, or specific combinations of these individual aspects.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive. The above description of example embodiments of theinvention has been presented for the purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinvention to the precise form described, and many modifications andvariations are possible in light of the teaching above.

A recitation of “a”, “an” or “the” is intended to mean “one or more”unless specifically indicated to the contrary. The use of “or” isintended to mean an “inclusive or,” and not an “exclusive or” unlessspecifically indicated to the contrary. Reference to a “first” componentdoes not necessarily require that a second component be provided.Moreover reference to a “first” or a “second” component does not limitthe referenced component to a particular location unless expresslystated. The terms “first” and “second” when used herein with referenceto elements or properties are simply to more clearly distinguish the twoor more elements or properties and unless stated otherwise are notintended to indicate order.

The terms “about” and “approximately equal” are used herein to modify anumerical value and indicate a defined range around that value. If “X”is the value, “about X” or “approximately equal to X” generallyindicates a value from 0.90X to 1.10X. Any reference to “about X”indicates at least the values X, 0.90X, 0.91X, 0.92X, 0.93X, 0.94X,0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X,1.06X, 1.07X, 1.08X, 1.09X, and 1.10X. Thus, “about X” is intended todisclose, e.g., “0.98X.” When “about” is applied to the beginning of anumerical range, it applies to both ends of the range. Thus, “from about6 to 8.5” is equivalent to “from about 6 to about 8.5.” When “about” isapplied to the first value of a set of values, it applies to all valuesin that set. Thus, “about 7, 9, or 11%” is equivalent to “about 7%,about 9%, or about 11%.”

All patents, patent applications, publications, and descriptionsmentioned herein are incorporated by reference in their entirety for allpurposes. None is admitted to be prior art.

What is claimed is:
 1. A method of analyzing a molecule, the methodcomprising: forming a membrane that divides a first reservoir from asecond reservoir, wherein the first reservoir has a first reservoirosmolarity, and wherein the second reservoir has a second reservoirosmolarity; flowing an electrolyte solution to the first reservoir,wherein the electrolyte solution has an electrolyte solution osmolaritythat differs from the first reservoir osmolarity, thereby making a firstchange to a ratio of the first reservoir osmolarity to the secondreservoir osmolarity; and applying a voltage across the membrane,wherein the membrane includes a nanopore, and wherein the voltage causesa net transfer of ions between the first reservoir and the secondreservoir, thereby making a second change to the ratio of the firstreservoir osmolarity to the second reservoir osmolarity, wherein thefirst change to the ratio and the second change to the ratiosubstantially counterbalance each other.
 2. The method of claim 1,wherein a volume of the first reservoir has an initial value beforeflowing the electrolyte solution, wherein the first change the ratio ofthe first reservoir osmolarity to the second reservoir osmolarity causeswater to flow across the membrane, thereby causing a first change to thevolume of the first reservoir, wherein the second change to the ratio ofthe first reservoir osmolarity to the second reservoir osmolarity causeswater to flow across the membrane, thereby causing a second change tothe volume of the first reservoir, and wherein the first change to thevolume of the first reservoir substantially counterbalances the secondchange to the volume of the first reservoir.
 3. The method of claim 1,wherein the net transfer of ions between the first reservoir and thesecond reservoir comprises a net efflux of ions from the secondreservoir to the first reservoir.
 4. The method of claim 3, wherein thenet efflux of ions from the second reservoir to the first reservoirincreases the ratio of the first reservoir osmolarity to the secondreservoir osmolarity, and wherein flowing the electrolyte solution tothe first reservoir decreases the ratio of the first reservoirosmolarity to the second reservoir osmolarity.
 5. The method of claim 4,wherein the electrolyte solution osmolarity is lower than the secondreservoir osmolarity before the electrolyte solution is flowed to thefirst reservoir.
 6. The method of claim 5, further comprising:progressively reducing the electrolyte solution osmolarity from aninitial electrolyte solution osmolarity to a final electrolyte solutionosmolarity to make the first change to the ratio of the first reservoirosmolarity to the second reservoir osmolarity.
 7. The method of claim 1,wherein the net transfer of ions between the first reservoir and thesecond reservoir comprises a net influx of ions into the secondreservoir from the first reservoir.
 8. The method of claim 7, whereinthe net influx of ions into the second reservoir from the firstreservoir decreases the ratio of the first reservoir osmolarity to thesecond reservoir osmolarity, and wherein flowing the electrolytesolution to the first reservoir increases the ratio of the firstreservoir osmolarity to the second reservoir osmolarity.
 9. The methodof claim 8, wherein the electrolyte solution osmolarity is higher thanthe second reservoir osmolarity before the electrolyte solution isflowed to the first reservoir.
 10. The method of claim 9, furthercomprising: progressively increasing the electrolyte solution from aninitial electrolyte solution osmolarity to a final electrolyte solutionosmolarity to make the first change to the ratio of the first reservoirosmolarity to the second reservoir osmolarity.
 11. The method of claim1, further comprising: inserting the nanopore into the membrane beforethe electrolyte solution is flowed to the first reservoir.
 12. Themethod of claim 1, further comprising: inserting the nanopore into themembrane after the electrolyte solution is flowed to the firstreservoir.
 13. The method of claim 1, wherein the membrane spans acrossthe second reservoir, wherein the first reservoir is external to thesecond reservoir, wherein the first reservoir has a first reservoirvolume, wherein the second reservoir has a second reservoir volume, andwherein the first reservoir volume is larger than the second reservoirvolume.
 14. The method of claim 1, wherein the voltage applied acrossthe membrane is an alternating current voltage.
 15. A method ofanalyzing a molecule, the method comprising: flowing an electrolytesolution to a first reservoir having a first reservoir osmolarity andover a membrane that divides the first reservoir from a second reservoirhaving a second reservoir osmolarity, wherein the electrolyte solutionhas an electrolyte solution osmolarity that differs from the firstreservoir osmolarity, thereby making a first change to a ratio of thefirst reservoir osmolarity to the second reservoir osmolarity; andapplying a voltage across the membrane, wherein the membrane includes ananopore, and wherein the voltage causes a net transfer of ions betweenthe first reservoir and the second reservoir, thereby making a secondchange to the ratio of the first reservoir osmolarity to the secondreservoir osmolarity, wherein the second change to the ratio at leastpartially offsets the first change to the ratio.
 16. The method of claim15, wherein the net transfer of ions between the first reservoir and thesecond reservoir comprises a net efflux of ions from the secondreservoir to the first reservoir.
 17. The method of claim 16, whereinthe net efflux of ions from the second reservoir to the first reservoirincreases the ratio of the first reservoir osmolarity to the secondreservoir osmolarity, and wherein flowing the electrolyte solution tothe first reservoir decreases the ratio of the first reservoirosmolarity to the second reservoir osmolarity.